Protein kinase C is a recently discovered enzyme found to phosphorylate basic proteins such as protamine and histone and other proteins. The phosphorylation of a protein may cause a functional change, which is generally a decrease or increase in enzymatic activity. Although protein kinase C is found in highest concentrations in the brain and spleen, it exhibits neither tissue nor species specificity.
Unlike other protein kinases, activation of protein kinase C is not dependent upon cyclic nucleotides, but activation does require phospholipid, diacylglycerol and calcium for maximum activity in vitro. Although diacylglycerol typically is not present in sufficiently high concentrations in cells to activate protein kinase C, it is transiently generated by the breakdown of phosphatidylinositol which is cleaved by phopholipase C, which in turn is activated when cells are stimulated by extra-cellular messengers. Such messengers include histamine, mitogens, growth factors, lymphokines, epinephrine, acetylcholine, vasopressin, gastrin and thrombin. With the breakdown of phosphatidylinositol, an intracellular increase in calcium (Ca.sup.2+) occurs. The calcium acts not only independently of and upon, but also synergistically with protein kinase C to produce many of the same physiological responses initiated by the increase in phosphatidylinositol turnover by extra-cellular messengers.
It has recently been found that tumor promoting phorbol diesters can substitute for diacylglycerol as activators for protein kinase C both in vitro and in vivo. The activities of phorbol diesters are mediated through protein kinase C which in actuality serves as a receptor for the phorbol diester. Phorbol diesters are tumor promoters, and induce tumor formation when administered with a carcinogen. When administered to cells in culture, phorbol diesters may induce transformation-like phenotypes or differentiation. It is known that cells possess high affinity, saturable, stereospecific receptors for phorbol diesters. The foregoing raises the possibility that protein kinase C, already known to be involved in normal cellular responses to external physiological signals, likely plays a role in the activities of tumor promoters, such as phorbol diesters. Thus, increased knowledge concerning the enzymology of protein kinase C may lead to a greater understanding of the actions of tumor promoters.
As mentioned above, protein kinase C phosphorylates a variety of basic proteins and a number of protein substrates. The following parameters are important in determining the "goodness" of a phosphorylation carried by protein kinase C: (a) phosphorylation is stoichiometric, i.e., at least 1 mole of phosphate is incorporated per mole of substrate; (b) one, two or three discrete sites on the protein molecule are phosphorylated, each in a stoichiometric manner; and, (c) the reaction is relatively rapid, usually completed within seconds or minutes, and occasionally as long as one hour. In efficient phosphorylation by protein kinase C, the Michaelis-Menten constant (K.sub.m) should be in the micromolar range and the maximum reaction rate ("V.sub.max ") should be between about 5 and 50 micromoles ("umole") PO.sub.4 transferred per min. per mg of protein.
The various in vitro phosphorylations catalized by protein kinase C include phosphorylation of guanylate cyclase, which causes an increase in its activity. The tumor-promoter TPA (12-0-tetradecanoylphorbol-13-acetate) also increases both guanylate cyclase activity, and cyclic GMP levels when added to lymphocytes. Thus it appears that protein kinase C phosphorylation of guanylate cyclase in vitro and in the cell increases this enzyme's activity. Protein kinase C cataylizes phosphorylation of 3-hydroxy-3-methylglutonyl coenzyme A reductase (HMG-CoA reductase), which is the rate-limiting enzyme in the cholesterol biosynthesis pathway. The protein kinase C-induced phosphorylation inactivates HMG-CoA reductase. Protein kinase C activates through phosphorylation tyrosine hydroxylase, which is a rate-limiting enzyme in the biosynthesis of catecholamines. The site of phosphorylation of the substrate appears to be the same to that of cAMP-dependent protein kinase. Protein kinase C also phosphorylates smooth muscle heavy meromyosin, thereby decreasing its actin-activated Mg-ATPase activity. In this situation, the site of phosphorylation by protein kinase C is different than the site of phosphorylation by cAMP-dependent protein kinase. Addition of protein kinase C to cardiac sarcoplasmic reticulum causes phosphorylation of phospholambin and Ca.sup.2+ uptake with the site of phosphorylation being different than that of cAMP-dependent protein kinase and calmodulin-dependent protein kinase. Further, protein kinase C phosphorylates glycogen synthetase, thereby inactivating this rate-limiting enzyme for glycogen synthesis. There are many sites phosphorylated on glycogen synthetase by a variety of protein kinases, including cAMP-dependent protein kinase. However, relative to these other protein kinases, phosphorylation by protein kinase C is uniquely selective.
Protein kinase C has been found to phosphorylate the basic nuclear proteins of the high-mobility group (HMG), protein 14 and 17. Protein kinase C phosphorylates a single site on HMB 17, Gly-Arg-Arg-Ser (P) Ala-Arg-Leu-Ser-Ala-Lys and also a single site on HMG 14, Pro-Lys-Arg-Arg-Ser (P) Ala-Arg-Leu (the "(P)" indicating the site of phosphorylation). cAMP-dependent protein kinase phosphorylates these basic nuclear proteins but not at the same locations phosphorylated by protein kinase C.
Perhaps the most extensively-studied receptor for a polypeptide growth factor is the epidermal growth factor (EGF) receptor. The receptor is itself a tyrosine protein kinase, a transmembrane protein with a domain that binds external EGF and a cytoplasmic domain that encodes a protein kinase activity. When bound by EGF, this kinase undergoes an intramolecular tyrosine autophosphorylation which causes partial activation of enzyme. Protein kinase C has been found to phosphorylate the EGF receptor thereby causing an increase in serine and threonine phosphorylation, but a decrease in tyrosine phosphorylation. Hunter et al., 1984 Nature 311: 480-483, have identified the major site of phosphorylation of the EGF receptor by protein kinase C through the use of a peptide composed of the following 24 amino acids corresponding to residues 643-646 of the receptor: Trp-Leu-Arg-Arg-Arg-His-Ile-Val-Arg-Lys-Arg-Thr (P) Leu-Arg-Arg-Leu-Leu-Gln-Glu-Arg-Glu-Leu-Val-Glu. Hunter et al. used this synthetic peptide as a substrate for protein kinase C. The relatively long length of this peptide indicates that its secondary structure most likely is a factor in its ability to function as an efficient substrate for protein kinase C.